Bottom Line:
A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications.Therefore, a more reliable ES cell culture technique is required.We used feeder-free and serum-free media without LIF.

Background: Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required.

Methodology/principal findings: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF.

pone-0006343-g001: Morphological changes of cultured mouse ES cells on Day 3 and 7.All the cells became oval cell shapes and flattened, the phenotype of differentiated ES cells in group 1G (a, b). The cells of group CL showed the formation of cell spheres (c, d).

Mentions:
Group 1G cells (ES cells in normal 1G environment) were morphologically different from general ES cells. They included forms similar to those of differentiated cells (Fig. 1a,b). On the other hand, group CL cells (ES cells in simulated microgravity environment) formed many small spheres after three days of culture (Fig. 1c). These floating spheres grew bigger during the culture period (Fig. 1d). After seven days of culture, the number of cells in group CL was about eight-times the number for group 1G. We tested these spheres to determine if they were indeed ES cells, and not embryonic body cells. Alkaline phosphatase (ALP) staining results indicated that the mouse ES cells had grown in group CL without the usual support of LIF or the serum and feeder layer and were maintained in an undifferentiated state (Fig. 2a,b). After three days of culture, Sox2 expression decreased in the cells of group 1G and Oct-4 and Nanog expressions decreased by Day 7 (Fig.2c). Sphere-forming cells in group CL, however, maintained the expression of these undifferentiated markers. Moreover, we confirmed the pluripotency of cells cultured under simulated microgravity for seven days. Teratomas were only generated by subcutaneously injecting group CL cells into C57BL/6 mice. The teratoma contains a mixture of mature and immature elements derived from the ectoderm, endoderm and mesoderm in a haphazard distribution (Fig. 3).

pone-0006343-g001: Morphological changes of cultured mouse ES cells on Day 3 and 7.All the cells became oval cell shapes and flattened, the phenotype of differentiated ES cells in group 1G (a, b). The cells of group CL showed the formation of cell spheres (c, d).

Mentions:
Group 1G cells (ES cells in normal 1G environment) were morphologically different from general ES cells. They included forms similar to those of differentiated cells (Fig. 1a,b). On the other hand, group CL cells (ES cells in simulated microgravity environment) formed many small spheres after three days of culture (Fig. 1c). These floating spheres grew bigger during the culture period (Fig. 1d). After seven days of culture, the number of cells in group CL was about eight-times the number for group 1G. We tested these spheres to determine if they were indeed ES cells, and not embryonic body cells. Alkaline phosphatase (ALP) staining results indicated that the mouse ES cells had grown in group CL without the usual support of LIF or the serum and feeder layer and were maintained in an undifferentiated state (Fig. 2a,b). After three days of culture, Sox2 expression decreased in the cells of group 1G and Oct-4 and Nanog expressions decreased by Day 7 (Fig.2c). Sphere-forming cells in group CL, however, maintained the expression of these undifferentiated markers. Moreover, we confirmed the pluripotency of cells cultured under simulated microgravity for seven days. Teratomas were only generated by subcutaneously injecting group CL cells into C57BL/6 mice. The teratoma contains a mixture of mature and immature elements derived from the ectoderm, endoderm and mesoderm in a haphazard distribution (Fig. 3).

Bottom Line:
A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications.Therefore, a more reliable ES cell culture technique is required.We used feeder-free and serum-free media without LIF.

Background: Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required.

Methodology/principal findings: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF.